This proposal aims at elucidating the functional mechanisms of a selected group of synthetases and coupled-ATPases. With synthetases, the mechanism of phosphonyl transfer will be studied with stereospecifically labeled (18O)-ATP. Specifically, the catalysis of bridge nonbridge oxygen scrambling will be assessed as a method for detecting enzyme-bound ATP cleavage products. This principle will be applied to GTP-dependent synthetases using (17O)-GTP and 17O-nuclease magnetic resonance. The absolute stereochemical course of ATP cleavage to AMP + PPi will be studied with the diastereoisomers of ATP (alpha-thiophosphate). With ATPases, the mechanism of "intermediate oxygen exchange" will be studied with the ATP oxygen scrambling technique.